Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes

Prerana Gogoi; Momoko Shiozaki; Eric Gouaux

doi:10.1371/journal.pone.0271799

Peer Review History

Original SubmissionJanuary 25, 2022
28 Mar 2022 Decision Letter - Sang H Lee, Editor PONE-D-22-00522Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomesPLOS ONE Dear Dr. Gouaux, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please provide a Link, in the Materials and Methods section, to the protocol.io component, using the digital object identifier (DOI) and format provided by protocols.io, as specified in the submissions requirements for Lab Protocols at https://journals.plos.org/plosone/s/submission-guidelines#loc-lab-protocols. Please submit your revised manuscript by May 12 2022 11:59PM. 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To comply with PLOS ONE submissions requirements, please provide the Protocols.io DOI in the Methods section of the manuscript using this format: “The protocol described in this peer-reviewed article is published on protocols.io, https://dx.doi.org/10.17504/protocols.io[........] and is included for printing as supporting information file 1 with this article.” Please also provide the Protocols.io DOI in the “Protocol DOI” field of the submission form (via “Edit Submission”). For more information, please see our submission guidelines: https://journals.plos.org/plosone/s/submission-guidelines#loc-guidelines-for-specific-study-types [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes Reviewer #2: No ******** 2. Has the protocol been described in sufficient detail? Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes Reviewer #2: Partly ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: No Reviewer #2: No ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: N/A ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript by Gogoi et al. described the workflow for the preparation of glutamatergic synaptosomes for cryo-electron tomography (cryo-ET) analysis. To identify the glutamatergic synaptosomes, the authors took advantage of the knock-in mouse brain in which presynaptic vGLUT1-mVenus is expressed and the GluA2 specific antibody fragment tagged with mCherry (15F1 Fab-mCherry). After density gradient centrifugation, the authors identified glutamatergic synaptosomes by cryo-confocal fluorescence microscopy, which visualizes the presynaptic vGLUT1-mVenus and postsynaptic AMPAR-15F1 Fab-mCherry-positive synaptosomes. Subsequently, cryo-FIB milling was used for rendering lamellae for cryo-ET analysis. Although cryo-ET studies are now topical and timely issues and the manuscript is clearly written, the actual cryo-ET data (i.e., cryo-ET imaging of this synaptosomes) is missing. So, the readers cannot evaluate this protocol. Major comments 1. The authors should add the representative cryo-ET images of glutamatergic synaptosomes acquired by this method. 2. The authors should cite the two key references in the cryo-ET studies of synapses and discuss them. (1) Rubén Fernández-Busnadiego. Cryo-Electron Tomography of the Mammalian Synapse. Methods Mol Biol. 2018;1847:217-224. doi: 10.1007/978-1-4939-8719-1_16. (2) Chang-Lu Tao et al. Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy. J Neurosci 2018 Feb 7;38(6):1493-1510.doi: 10.1523/JNEUROSCI.1548-17.2017. Minor comment The authors should spell out “FSEC” in the line 111 and “Pt GIS” in the lien 161. Reviewer #2: This brief protocol describes a synaptoneurosome preparation for cryoET studies. The data would be better suited as part of a research article, at present it is unclear whether this workflow will permit structural characterisation of synaptic receptor complexes. Other comments 1. It would be helpful to present a workflow schematic in the main text figure, as it is currently not described with sufficient clarity. 2. In the “Cryo-FIB milling of glutamatergic synaptosomes” section, fluorescent light microscope images were used for the guidance of FIB milling. An overlay image of FLM and SEM would be needed to demonstrate the alignment. 3. In main text line 138, the authors mentioned filtering of synaptosomes by using a thermobarrel extruder, was this sample used for the following EM grids preparation and FIB milling? Please clarify 4. a TEM image of a representative lamella and an its overlay with the corresponding light microscope image should be added to figure 5. 5. The authors state that by following their procedure the final lamella can reach 100-200 nm thickness, a section of a reconstructed tomogram or other evidence should be added to support this statement. 6. In S1 Fig, the authors mention the existence of other components in density gradient fractions ( “myelin & membranes and extrasynaptosomal mitochondria”). Western blot or other experimental evidence is needed to support the identification of those components. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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Revision 1
12 May 2022 Author Response Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript by Gogoi et al. described the workflow for the preparation of glutamatergic synaptosomes for cryo-electron tomography (cryo-ET) analysis. To identify the glutamatergic synaptosomes, the authors took advantage of the knock-in mouse brain in which presynaptic vGLUT1-mVenus is expressed and the GluA2 specific antibody fragment tagged with mCherry (15F1 Fab-mCherry). After density gradient centrifugation, the authors identified glutamatergic synaptosomes by cryo-confocal fluorescence microscopy, which visualizes the presynaptic vGLUT1-mVenus and postsynaptic AMPAR-15F1 Fab-mCherry-positive synaptosomes. Subsequently, cryo-FIB milling was used for rendering lamellae for cryo-ET analysis. Although cryo-ET studies are now topical and timely issues and the manuscript is clearly written, the actual cryo-ET data (i.e., cryo-ET imaging of this synaptosomes) is missing. So, the readers cannot evaluate this protocol. Major comments 1. The authors should add the representative cryo-ET images of glutamatergic synaptosomes acquired by this method. We thank the reviewer for the suggestion and we have added a reconstructed tomogram of synaptosomes acquired by this method to the revised version of the manuscript. 2. The authors should cite the two key references in the cryo-ET studies of synapses and discuss them. (1) Rubén Fernández-Busnadiego. Cryo-Electron Tomography of the Mammalian Synapse. Methods Mol Biol. 2018;1847:217-224. doi: 10.1007/978-1-4939-8719-1_16. (2) Chang-Lu Tao et al. Differentiation and Characterization of Excitatory and Inhibitory Synapses by Cryo-electron Tomography and Correlative Microscopy. J Neurosci 2018 Feb 7;38(6):1493-1510.doi: 10.1523/JNEUROSCI.1548-17.2017. As suggested by the reviewer, both the references are discussed in the revised version of the manuscript as suggested by the reviewer. Minor comment The authors should spell out “FSEC” in the line 111 and “Pt GIS” in the lien 161. “FSEC” and “Pt GIS” have been spelled out in the revised version of the manuscript. Reviewer #2: This brief protocol describes a synaptoneurosome preparation for cryoET studies. The data would be better suited as part of a research article, at present it is unclear whether this workflow will permit structural characterisation of synaptic receptor complexes. Other comments 1. It would be helpful to present a workflow schematic in the main text figure, as it is currently not described with sufficient clarity. We thank the reviewer for the suggestion and we have incorporated a workflow schematic in the main text (Fig 1). 2. In the “Cryo-FIB milling of glutamatergic synaptosomes” section, fluorescent light microscope images were used for the guidance of FIB milling. An overlay image of FLM and SEM would be needed to demonstrate the alignment. Although fluorescence light images were used to guide the FIB-milling, we were unable to discern synaptosome-like features in the SEM images, simply due to the constraints of the imaging mode. Thus, we do not believe that an overlay of the FLM and SEM images would constructively augment the manuscript. What we did do, however, was to perform fluorescence imaging on the FIB-milled lamella, to confirm that our FLM guided milling was successful, as evidenced by the presence of adjacent or overlapping red and green fluorescence (Fig 6). 3. In main text line 138, the authors mentioned filtering of synaptosomes by using a thermobarrel extruder, was this sample used for the following EM grids preparation and FIB milling? Please clarify In the revised version of the manuscript, we have mentioned that the filtered synaptosomes were used for the preparation of EM grids. 4. a TEM image of a representative lamella and an its overlay with the corresponding light microscope image should be added to figure 5. TEM images were acquired on the lamella for which overlapping green and red fluorescence signal were detected in the cryo-LSM. However, we have not employed any tools or other ways to overlay the images. 5. The authors state that by following their procedure the final lamella can reach 100-200 nm thickness, a section of a reconstructed tomogram or other evidence should be added to support this statement. The thickness of the lamella was measured during cryo FIB milling using the AutoTEM 2.0 software and monitored using SEM. To further support the preparation of successful synaptosome lamellae, we have added a reconstructed tomogram to the revised version of the manuscript. 6. In S1 Fig, the authors mention the existence of other components in density gradient fractions ( “myelin & membranes and extrasynaptosomal mitochondria”). Western blot or other experimental evidence is needed to support the identification of those components. In this study, the steps for density gradient separation of synaptosomes from other components has been performed and similar results were reproduced as described in previous studies (Gray and Whittaker, 1962; Cotman and Matthews, 1971; Dunkley et al., 2008). All these studies have been cited appropriately (S1 Fig legend). Given the extensive prior documentation, we respectively assert that further analysis is not a productive use of time or resources. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0271799.r002
8 Jul 2022 Decision Letter - Sang H Lee, Editor Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes PONE-D-22-00522R1 Dear Dr. Gouaux, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Sang H Lee, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature? Reviewer #1: Yes ******** 2. Has the protocol been described in sufficient detail? Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible. Reviewer #1: Yes ****** 3. Does the protocol describe a validated method? The manuscript must demonstrate that the protocol achieves its intended purpose: either by containing appropriate validation data, or referencing at least one original research article in which the protocol was used to generate data. Reviewer #1: Yes ****** 4. If the manuscript contains new data, have the authors made this data fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the article presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please highlight any specific errors that need correcting in the box below. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors suitably responded to all my comments. Now, the paper will provide readers with a useful workflow for the preparation glutamatergic synaptosomes for Cryo-ET analysis. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0271799.r003
Formally Accepted
4 Aug 2022 Acceptance Letter - Sang H Lee, Editor PONE-D-22-00522R1 Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes Dear Dr. Gouaux: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Sang H Lee Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271799.r004

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